Genetic Evidence for the Persistence of the Critically Endangered Sierra Nevada Red Fox in California

Conserv Genet DOI 10.1007/s10592-006-9265-z

ORIGINAL PAPER

Genetic evidence for the persistence of the critically endangered Sierra Nevada red fox in California

John D. Perrine Æ John P. Pollinger Æ Benjamin N. Sacks Æ Reginald H. Barrett Æ Robert K. Wayne

Received: 10 July 2006 / Accepted: 20 November 2006 Springer Science+Business Media B.V. 2007

Abstract California is home to both the native state- high-elevation populations (average pairwise FST = threatened Sierra Nevada red fox (Vulpes vulpes ne- 0.06), but these populations differed substantially from cator), which historically inhabited high elevations of all modern and historic lowland populations (average the Sierra Nevada and Cascade mountains, and to pairwise FST = 0.52). Among lowland populations, the multiple low-elevation red fox populations thought to historic and modern Sacramento Valley populations be of exotic origin. During the past few decades the were not signiﬁcantly differentiated from one another lowland populations have dramatically expanded their (FST = –0.06), but differed signiﬁcantly from recently distribution, and possibly moved into the historic range founded populations in the San Francisco Bay region of the native high-elevation fox. To determine whether and in southern California (average pairwise FST = the native red fox persists in its historic range in Cali- 0.42). Analysis of molecular variance indicated that 3 fornia, we compared mitochondrial cytochrome-b population groupings (mountain, Sacramento Valley, haplotypes of the only currently-known high-elevation and other lowland regions) explained 45% of molecu- population (n = 9 individuals) to samples from 3 lar variance (FCT = 0.45) whereas only 4.5% of the modern lowland populations (n = 35) and historic variance was partitioned among populations within

(1911–1941) high-elevation (n = 22) and lowland these groupings (FSC = 0.08). These findings provide (n = 7) populations. We found no significant popula- strong evidence that the native Sierra Nevada red fox tion differentiation among the modern and historic has persisted in northern California. However, all nine samples from this population had the same haplotype, suggesting that several historic haplotypes may have J. D. Perrine (&) Á R. H. Barrett become lost. Unidentified barriers have apparently Department of Environmental Sciences, Policy, and prevented gene flow from the Sacramento Valley Management, University of California, Berkeley, CA 94720-3110, USA population to other eastern or southern populations in e-mail: [email protected] California. Future studies involving nuclear markers are needed to assess the origin of the Sierra Nevada J. P. Pollinger Á R. K. Wayne red fox and to quantify levels of nuclear gene flow. Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095-1606, USA Keywords Gene flow Á Population structure Á B. N. Sacks Sierra Nevada red fox Á Threatened species Á Vulpes Canid Diversity and Conservation Project, Veterinary vulpes necator Genetics Laboratory, University of California, Davis, CA 95616-8744, USA

Present address: Introduction J. D. Perrine Museum of Vertebrate Zoology, University of California, 3101 Valley Life Sciences Building, Berkeley, CA 94720- California is currently home to both native and exotic 3160, USA populations of red fox (Vulpes vulpes). The native

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Sierra Nevada red fox (V. v. necator) is restricted to particularly valuable for comparing modern and his- subalpine habitats above 1525 m (5000 ft) in the Sierra toric levels of genetic diversity within populations that Nevada and Cascade mountain ranges of California have subsequently become rare or endangered. This (Grinnell et al. 1937; Schempf and White 1977). His- approach has been applied across a wide range of torically the Sierra Nevada red fox existed at low animal taxa and conservation questions, such as densities throughout its range (Grinnell et al. 1937), investigating bottlenecks in the greater prairie chicken but an apparently precipitous population decline led (Tympanuchus cupido; Bouzat et al. 1998), northern state wildlife officials to prohibit commercial trapping elephant seal (Mirounga angustirostris; Weber et al. in 1974 and to list the subspecies as State Threatened in 2000), Yellowstone grizzly bear (Ursus arctos; Miller 1980 (Gould 1980; Lewis et al. 1999). It is currently and Waits 2003), and black-footed ferret (Mustela considered ‘‘extremely endangered’’ and its population nigripes; Wisely et al. 2002); discerning the taxonomic size, extent, and trend are unknown (CDFG 1996, affiliation of the Dawson caribou (Rangifer tarandus 2004). dawsoni; Byun et al. 2002), red wolf (Canis rufus; Roy The other red fox populations in California inhabit et al. 1996), and Uele River gorilla (Gorilla gorilla the lowland areas (<1066 m) and likely consist of uellensis; Hofreiter et al. 2003); and quantifying the individuals from multiple unidentified source popula- genetic impact of recent forest fragmentation on red tions (Grinnell et al. 1937; Roest 1977; CDFG 1999; squirrels (Sciurus vulgaris; Hale et al. 2001). The ap- Fitzpatrick 1999; Lewis et al. 1999). Lowland foxes proach has proven particularly useful for examining were first recorded in the 1880s from the plains near temporal changes in population structure, such as in the Sutter Buttes in the Sacramento Valley and their the Panamint kangaroo rat (Dipodomys panamintinus; origin is unknown, although some individuals may have Thomas et al. 1990), bearded vulture (Gypaetus barb- been transported from elsewhere in North America for atus; Gautschi et al. 2000), northeastern beach tiger sport hunting or rodent control (Grinnell et al. 1937; beetle (Cicindela dorsalis dorsalis; Goldstein and Roest 1977; Lewis et al. 1999). Low elevation red foxes DeSalle 2003), North Sea cod (Gadus morhua; remained restricted to the Sacramento Valley through Hutchinson et al. 2003), and adonis blue butterfly the early decades of the 20th century (Roest 1977; (Polyommatus bellargus; Harper et al. 2006). Lewis et al. 1999). By the 1990s, the range of the Until recently, analysis of historic and recent Cali- ‘‘lowland red fox’’ had expanded dramatically, fornia red foxes was hindered not by the lack of mu- extending throughout the Sacramento and San Joaquin seum specimens but rather by the absence of mountain Valleys to the Sierra Nevada foothills and in various fox specimens collected after 1950 (Fig. 1). A recent coastal locations from San Francisco to San Diego ecological study of the red foxes in the Lassen Peak (Lewis et al. 1999). However, it is unclear to what ex- region of northern California (1900–3150 m elevation) tent the new lowland populations (i.e., those outside enabled the collection of genetic samples for this pur- the Sacramento Valley) arose from the original Sac- pose (Perrine 2005). The Lassen area was historically a ramento Valley population; some of them likely derive main population center for the Sierra Nevada red fox from additional introductions from outside of Califor- (Grinnell et al. 1937; Schempf and White 1977) and is nia after 1950 (CDFG 1999; Fitzpatrick 1999; Lewis currently the only known montane red fox population et al. 1999). Moreover, it is unknown whether lowland in the state (Perrine 2005). However, the Lassen area red foxes have expanded into the historic range of the may also be highly vulnerable to colonization by low- native mountain red fox (Lewis et al. 1995). Morpho- land red foxes from the Sacramento Valley <70 km logical characteristics are insufficient to confidently away, which is within the potential dispersal radius of conclude whether an individual red fox originated from red fox (Larivie`re and Pasitschniak-Arts 1996). Fur- either the native or exotic populations (Roest 1977). A thermore, escapees from several red fox fur farms in genetic comparison of present-day and historic red the Lassen region during the 1940s and 1950s could foxes is needed to ascertain the ancestry of these have become naturalized in the surrounding area populations so that appropriate management actions (Lewis et al. 1995, 1999). can be taken (Kucera 1995, 1999; Lewis et al. 1995; Our primary objective in this study was to test the Aubry 1997). hypothesis that the Lassen red foxes are descendants Advances in DNA extraction techniques have en- of the native mountain population rather than exotic abled the collection of genetic data from museum colonists. Our secondary objectives were to quantify specimens for use in evolutionary and population ge- the diversity and distribution of mitochondrial netic studies (Paä¨bo 1989; Cooper 1994; Hummel 2003; haplotypes within California, especially those unique Paä¨bo et al. 2004). Museum specimens have proven to the mountain or lowland populations, and to obtain

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3500

3000

2500 Bay Area Cascades 2000 Southern California 1500 Sierra Nevada Sacramento Valley Elevation (m) 1000

500

0 1900 1920 1940 1960 1980 2000 Year

Fig. 1 Distribution of red fox specimens according to collection were collected near these boundary lines, making it highly year and elevation. Horizontal dashed line at 1066 m differen- unlikely that any were misclassified. Note the lack of modern tiates ‘‘lowland’’ from ‘‘high-elevation’’; vertical dashed line at high-elevation specimens prior to 2000 1950 differentiates ‘‘historic’’ from ‘‘modern.’’ Few specimens a preliminary assessment of population structure Vertebrate Zoology at the University of California, within the state for use in determining sampling pri- Berkeley (n = 45), other university and municipal orities for future research. Analyses were based on a natural history collections in California (n = 22), and 354 base-pair region of the mitochondrial cyto- from several road-killed red foxes and one skeleton chrome-b gene sequenced from historic and modern collected in the Sacramento Valley (see Appendix foxes from mountain and lowland locations through- Table 4). Extractions were first attempted using out California. untanned hide or muscle, and if these were unavailable or did not yield usable cytochrome-b sequences, we used maxilloturbinal bones (Wisely et al. 2004b) as the Materials and methods source for DNA. We classified specimens based upon the elevation Samples and date of their collection (Fig. 1). Only specimens with unambiguous collection dates and localities were We obtained a total of 85 samples for genetic analyses, included in our analyses. In keeping with the opera- including five ear punches and nine feces collected tional criterion used by the California Department of during the Lassen Peak study (1998–2002; Perrine Fish and Game (e.g., Lewis et al. 1993), specimens 2005). The nine feces, selected from a total of 227 red collected above 1066 m (3500 ft) were considered fox feces collected during the study, were considered ‘‘mountain’’ and those below 1066 m were ‘‘lowland.’’ likely to represent additional individuals because they Similarly, those collected prior to 1950 were considered were found outside the home ranges of the five col- ‘‘historic’’ and those collected after 1950 were consid- lared foxes that provided the ear punches. Four of the ered ‘‘modern.’’ We chose 1950 as a temporal bound- nine fecal samples were included in our analysis after ary based upon a natural separation in the collection being reliably differentiated from one another and times of the available museum specimens and because from the five captured individuals by microsatellite the range of the lowland red fox had not expanded analysis (B. Sacks, unpublished data). Specifically, beyond the Sacramento Valley by this date (Lewis these samples were genotyped twice at 12 to 14 mi- et al. 1999). In total, comparisons were based on 7 crosatellite loci and differed by an average of 15 alleles sample groupings (hereafter, ‘‘populations’’): 3 from (>50%; range 5–19 alleles). Because allelic dropout the mountains (Historic Cascades, Historic Sierra Ne- could have influenced these comparisons, we only in- vada, Modern Cascades) and 4 from the lowlands cluded individuals that were differentiated from all (Historic Sacramento Valley, Modern Sacramento others at both allelic positions of at least one locus. The Valley, San Francisco Bay Area, Southern California) nine specimens from the Lassen Peak region likely (Fig. 2). San Francisco Bay Area (hereafter, ‘‘Bay represent a significant proportion of this highly local- Area’’) and Southern California populations did ized population (Perrine 2005). Specimens from not exist prior to 1950 and are therefore represented other populations were obtained from the Museum of solely as modern populations. All specimens from the

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Fig. 2 Distribution of red fox specimens and 7 putative Sierra Nevada red fox is unknown and is therefore assumed to be populations in California, relative to the range of the native the same as its historic distribution. CS = Cascades; SN = Sierra Sierra Nevada red fox (stippling) and the lowland red fox Nevada; SV = Sacramento Valley; BA = Bay Area; SC = South- (diagonal lines). Note the increase in the lowland red fox’s range ern California. Distributions based upon Grinnell et al. (1937) from the 1930s to the 1990s. The current distribution of the and Lewis et al. (1999)

Modern Cascades population were obtained from the followed by an additional 300 lg of proteinase K and Lassen Peak region. No modern specimens were 24 additional h at 56C. Samples were then centrifuged available from the Sierra Nevada Mountains. and 1 ml of the supernatant was transferred to a 10 ml In part of its geographic range in California, the tube containing 5 ml of Qiagen Buffer PB. The DNA lowland red fox overlaps in distribution with a con- was then bound, washed and resuspended in 50 llof genor, the kit fox (Vulpes macrotis; Hall 1981). How- TE buffer using the Qiagen Qiaquick PCR Puriﬁcation ever, V. vulpes and V. macrotis are unlikely to Kit. A negative control was run with each set of hybridize as they have dramatically different chromo- extractions to detect possible contamination. some numbers (2n = 48 and 36, respectively) and A 354 bp sequence of cytochrome-b was isolated chromosome morphology (Wayne et al. 1987) and using primers RF14724 (5¢-CAACTATAAGAACAT- genetically are not closely related (Lindblad-Toh et al. TAATGACC-3¢) and RF15149 (5¢-CTCAGAATGA- 2005). Cytochrome-b and other protein coding se- TATTTGTCCTC-3¢; 441 bp PCR product), modiﬁed quences differ by over 8% between the two species from L14724 and H15149, respectively (Irwin et al. (Wayne et al. 1997). 1991). Because the nasal turbinate samples were often degraded with DNA fragment lengths potentially Laboratory procedures shorter than the desired products, a set of shorter overlapping PCR products was used to generate the Hide and muscle samples were cut into 100–200 mg same DNA sequence: RF14724-RFCYTB3R and pieces, diced with a sterile blade, then soaked in sterile RFCYTBBF-RF15149 (RFCYTB3R: 5¢-GAT- 1 · PBS solution for 24 h to dilute any preservatives GCTCCGTTTGCATGTATG-3¢; start position 263 of that may have been present. DNA was then extracted cytochrome-b, 263 bp PCR product, and RFCYTBBF: using a QIAamp minikit (Qiagen Incorporated, 5¢-CTGCCGAGACGTTAACTATGGCTG-3¢; start Valencia, CA) and the standard tissue extraction pro- position 224 of cytochrome-b, 218 bp PCR product). tocol (200 ll elution volume). DNA was extracted PCR reactions were 25 ll total volume and con- from nasal turbinate samples using the method of sisted of 2 ll of tissue derived DNA or 5 ll of fecal or Wandeler et al. (2003b) in a separate isolated and nasal turbinate derived DNA, 2.5 llof10· PCR dedicated facility for low-copy DNA samples. A 100– buffer, 2.0 ll of 10 mM dNTPs, 1.5 llof25mM

300 mg sample of bone fragments was chilled in liquid MgCl2,1ll each of 10 lM forward and reverse prim- nitrogen in a sterile vial for 2 min, then pulverized into ers, 0.3 llof5U/ll Taq and remainder water. PCR a fine powder using a UV- and bleach-sterilized mortar cycle conditions were 94C for 3 min; then 45 cycles of and pestle. The powder was decalcified for 72 h by 94C for 30 sec, 50C for 30 sec and 72C for 45 sec; suspension and agitation in 1.5 ml of 0.5 M EDTA. followed by 10 min at 72C. PCR products were run on Samples were then digested with 60 ll of 10% N-sar- agarose gels and extracted using Ultraclean 15 DNA cosyl and 600 lg of proteinase K for 24 h at 56C, purification kits (Mo Bio, Solana Beach, CA) or were

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purified using multiscreen PCR micro 96 plates (Mil- Arlequin to generate a matrix of pairwise FST esti- lipore Corporation, Billerica, MA). Dye terminator mates (Weir and Cockerham 1984) among all popula- sequencing reactions were performed for each PCR tions based upon haplotype frequencies. Pairwise F ST product for each primer using Beckman DTCS re- were also estimated to incorporate pairwise differences agents and products were sequenced in both directions between haplotypes (Nei and Li 1979). Significance (a on a Beckman CEQ2000XL capillary sequencer (Ful- = 0.05) was calculated using 1000 permutations and lerton, CA) or using Applied Biosystems reagents with then corrected for multiple tests via the sequential products sequenced on an ABI 3730 capillary se- Bonferroni method (Rice 1989). Haplotypes based on quencer (Applied Biosystems, Foster City, CA). Se- homologous sequences from 41 red foxes widely dis- quences were deposited in the EMBL/Genbank/DDBJ tributed throughout Europe (Frati et al. 1998; Gen- nucleotide database (Accession Nos. EF064207– bank Accession Nos. Z80957–Z80997) were used to EF064220). help elucidate nonnative ancestry among California red foxes. Although we did not necessarily expect the Data analysis exotic foxes in our study to have been introduced di- rectly from Europe, the species originated and evolved Due to the matrilineal inheritance of mtDNA, we in- primarily in Eurasia (Kurteń 1980), making a Euro- cluded only one specimen per litter whenever such pean sample a useful reference to identify foreign information was known. Within each population, hap- haplotypes. lotype and nucleotide diversity (Watterson 1975) were estimated using Arlequin 2.000 (Schneider et al. 2000). Relationships among haplotypes were described using Results a minimum spanning tree. To assess differentiation among population groupings, we conducted a series of We obtained unambiguous cytochrome-b sequences hierarchical analyses of molecular variance (AMOVA; from 75 of the 85 specimens (88.2%). To be conser- Excoffier et al. 1992) using Arlequin 2.000. These in- vative, specimens yielding only partial sequences were cluded one temporal analysis (historic vs. modern not included in the analyses. Sequences from two populations) and two spatial analyses: (1) mountain specimens were excluded because they were litter- (Historic Sierra Nevada, Historic Cascades, Modern mates of other specimens that amplified successfully Cascades) versus lowland (Historic Sacramento Valley, (Appendix Table 4). The 73 remaining sequences had Modern Sacramento Valley, Bay Area, Southern Cal- 17 variable sites (13 transitions, 4 transversions) in ifornia); and (2) mountain, Sacramento Valley (His- 354 bp and defined 14 haplotypes (Table 1; Fig. 3). toric and Modern), and other lowland populations Haplotypes differed from haplotype A by up to 6 (Bay Area, Southern California). Because differentia- substitutions. Three haplotypes (G, M, N) shared a tion among mountain populations was hypothesized to signature of three distinctive polymorphisms with the be low (i.e., if originating from a single population) European haplotypes (Fig. 3), whereas the remaining relative to that among lowland populations (potentially haplotypes differed by one or two substitutions from high due to multiple source populations), we also used haplotype A.

Table 1 Occurrence of 14 mitochondrial cytochrome-b haplotypes in three historic (pre-1950) and four modern (post-1950) California red fox populations Populationa n Haplotypes A CD EFGHIJKMNOP

Historic SN 18 13 1 1 – – – – – 2 – – – 1 – CS 431–––––––––––– SV71–5––––––––––1 Modern CS 99––––––––––––– SV 12 1 – 10 – – 1 – – – – – – – – BA 10 – – – – 6 1 1 – – – 1 1 – – SC 13 – – – 1 2 2 – 1 – 6 – 1 – – a SN = Sierra Nevada, CS = Cascades, SV = Sacramento Valley, BA = San Francisco Bay Area, SC = Southern California

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specimens and 15 of 42 (35.7%) lowland specimens, all from the Sacramento Valley. All three haplotypes with the European signature were restricted to lowland populations. Of the eight haplotypes that were unique to a single population, six were represented by a single individual. Haplotype K occurred only in the six foxes from Santa Barbara County within the Southern California population. All nine specimens in the Modern Cascades (i.e., Lassen Peak) population had haplotype A, which was also the most common haplotype in the Historic Cas- cades (75%) and Historic Sierra Nevada (72.2%) populations. Three haplotypes occurred in the Modern Sacramento Valley population but D was the most prevalent (83.3%). Despite the geographic proximity of the Sacramento Valley and the Bay Area, they shared only haplotype G, which was present in one sample from each population. The Bay Area and Southern California populations shared three haplotypes and these populations had the greatest haplotype and nucleotide diversity (Table 2). The temporal AMOVA allocated no signiﬁcant Fig. 3 Variable sites in the 354 bp region of the cytochrome-b proportion of the genetic variation to time period gene in red foxes from California (this study) and Europe (historic vs. modern) (FCT = –0.07; P = 0.64). The 2- (n = 41; Frati et al. 1998). Note that the 17 European haplotypes shared bases in positions 168, 174 or 219. California haplotypes group spatial AMOVA allocated 30% of the genetic G, M, and N also shared these bases, in contrast with other variation to that between the mountain and lowland California haplotypes groups of populations (FCT = 0.30; P = 0.03) and 20.9% of the variability among the populations to that within Only haplotypes A and D (differing by one substi- the 2 groups (F = 0.30; P<0.001). The 3-group spatial tution) occurred in both high-elevation and lowland SC AMOVA allocated 45% of the genetic variation to populations, and the remaining 12 haplotypes were that among the mountain, Sacramento Valley, and exclusive to either the mountains or the lowlands other lowland population groupings (F = 0.45; P = (Table 1; Fig. 4). Haplotype A accounted for 25 of 31 CT 0.01) and only 4.5% of the variability among popula- (80.6%) mountain specimens and two of 42 (4.8%) tions within groups (F = 0.08; P<0.001). Thus, the lowland specimens (both from the Sacramento Valley). SC 3-group model was a considerably better ﬁt to the data Haplotype D accounted for one of 31 (3.2%) mountain than the 2-group model. Pairwise FST and F ST values

P Table 2 Haplotype and nucleotide diversity associated with a C D 354-bp sequence of the mitochondrial cytochrome-b gene among seven populations J Populationa n Haplotype SD Nucleotide SD diversity diversity A G O N M Historic I SN 18 0.48 0.14 0.0018 0.0016 F CS 4 0.50 0.27 0.0028 0.0028 K SV 7 0.52 0.21 0.0016 0.0017 E H Modern CS 9 0 0 0 0 Fig. 4 Minimum spanning tree illustrating relationships among SV 12 0.32 0.16 0.0027 0.0022 14 cytochrome-b haplotypes. Circles indicating haplotypes are BA 10 0.67 0.16 0.0087 0.0056 shown in proportion to their frequency in the sample and color- SC 13 0.78 0.10 0.0100 0.0061 coded with respect to their origin in mountain (white), Sacramento Valley (gray), or other lowland (black) population. a SN = Sierra Nevada, CS = Cascades, SV = Sacramento Val- Lines indicate unsampled haplotypes ley, BA = San Francisco Bay Area, SC = Southern California

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Table 3 Pairwise FST and FST estimates among three historic (pre-1950) and four modern (post-1950) California red fox populations Historic SN Historic CS Historic SV Modern CS Modern SV Modern BA Modern SC

Historic SNa – –0.08 0.51* 0.00 0.45* 0.36* 0.27* Historic CS –0.10 – 0.51 0.22 0.41* 0.18 0.11 Historic SV 0.42* 0.42 – 0.75* –0.06 0.40* 0.32* Modern CS 0.06 0.22 0.73* – 0.54* 0.31* 0.21 Modern SV 0.54* 0.60* –0.06 0.80* – 0.40* 0.33* Modern BA 0.44* 0.39* 0.40* 0.65* 0.51* – 0.09 Modern SC 0.38* 0.31* 0.33* 0.56* 0.44* 0.18 –

Below diagonal measures are based solely on haplotype frequencies (FST); above diagonal estimates incorporate pairwise differences in sequence divergence (FST) a SN = Sierra Nevada, CS = Cascades, SV = Sacramento Valley, BA = San Francisco Bay Area, SC = Southern California * significant at a = 0.05 using sequential Bonferroni correction for multiple tests (Rice 1989) were consistent with these analyses, indicating no sig- provided sufficient resolution to preliminarily assess nificant differentiation among the historic and modern structure among California red fox populations. In fact, high-elevation populations, but significant and sub- the diversity of haplotypes in California was compa- stantial genetic differentiation between these popula- rable that observed for the Mediterranean Basin tions and the Sacramento Valley populations (Fig. 3; Frati et al. 1998). However, inferences based (Table 3). Likewise, the modern and historic Sacra- on mitochondrial DNA reflect only matrilineal history mento Valley populations were not significantly dif- and gene flow. Juvenile male red foxes are more likely ferent from one another, but both differed significantly to disperse than females and usually travel two to three from the other 2 modern lowland populations. times as far (20–30 km for males, 10–15 km for females), although occasional instances of both males and females dispersing >70 km (the distance between Discussion Lassen Peak and the Sacramento Valley) have been documented (Phillips et al. 1972; Storm et al. 1976; Our study assessed the genetic structure among Voigt 1987; Rosatte 2002). Therefore, levels of nuclear mountain and lowland red fox populations in Califor- gene flow may be higher than revealed by analysis of nia before and after a marked range increase by the mtDNA. With this caveat in mind, however, several lowland populations. High phylogenetic divergence important findings emerged from our study, as dis- was expected between the mountain and lowland cussed below. populations due to their different hypothesized evolutionary origins. The Sierra Nevada red fox, along with Population differentiation the Cascade and Rocky Mountain red foxes (V. v. cascadensis and V. v. macroura, respectively), likely Analysis of cytochrome-b haplotype frequencies found derived from a lineage isolated south of the continental no significant genetic differentiation between modern ice sheets during the Wisconsonian glaciation. When and historic populations within the range of the Sierra the glaciers retreated this southern refugial population Nevada red fox in California. All nine of the modern became isolated in the subalpine and boreal habitats of Cascades specimens from Lassen Peak had the haplo- the western mountain ranges (Aubry 1983). In con- type (A) that was the most abundant haplotype in the trast, the modern-day low-elevation red foxes in Cali- Cascades and Sierra Nevada populations in California fornia are widely believed to have originated from nearly a century earlier. The prominence of this hap- various populations throughout eastern and northern lotype in the mountain populations and its scarcity North America, which may include European lineages among the lowland populations is strong evidence that due to introductions in the 17th and 18th centuries a remnant of the native, state-threatened Sierra Ne- (Churcher 1959; Roest 1977; Aubry 1983; Lewis et al. vada red fox persists in the Lassen Peak region. The 1999; Kamler and Ballard 2002). lack of haplotype diversity within this modern popu- The cytochrome-b gene was chosen over the more lation is consistent with high levels of genetic drift and variable control region to make use of a number of loss of rare alleles as would be expected within small, previously published cytochrome-b sequences for isolated populations (Wright 1978), as the Lassen Peak comparison (Geffen et al. 1992; Frati et al. 1998). Even population appears to be (Perrine 2005). We cannot, with the small sample sizes available, cytochrome-b however, exclude the possibility that the Lassen Peak

123 Conserv Genet individuals were from a single family group, although northern or eastern North America, whose red fox the temporal and spatial breadth of the sample makes populations are thought to have derived from Eur- this unlikely. The low levels of haplotype and nucleo- asian lineages during the Pleistocene (Aubry 1983), tide diversity observed in all three mountain fox pop- and were then translocated to California. Although ulations are consistent with other species thought to one specimen (CSU-2589) from the modern Sacra- exist in refugial Sierra Nevada populations (e.g., mento Valley population had the European signature, Wisely et al. 2004a). The lack of genetic differentiation no other haplotypes were shared between the Sac- between California’s Cascade and Sierra Nevada pop- ramento Valley and the other lowland populations. ulations supports Grinnell et al.’s (1937) characteriza- The absence of haplotypes A and D in the San tion of these populations as the same subspecies, as Francisco Bay Area and Southern California popu- opposed to the original inclusion of the California lations indicates that these populations were not Cascades population with the more northerly Cascade founded by individuals dispersing from the Sacra- Range populations in V. v. cascadensis (Merriam 1900; mento Valley. Grinnell et al. 1930). Moreover, haplotype O, which Our finding that the red fox populations in the Bay occurred in one fox from the historic Sierra Nevada Area and Southern California exhibited relatively low population and which differs from haplotype A by a genetic differentiation is in agreement with a previ- single substitution, is the dominant haplotype in mod- ous analysis of three California coastal red fox pop- ern Cascade foxes from Washington (Perrine 2005), ulations using three microsatellite loci and a 240 bp suggesting little differentiation between these two portion of the mitochondrial control region (Fitzpat- currently recognized subspecies. rick 1999). Low FST estimates may indicate high gene In contrast to the close relationships among moun- flow, but can also arise due to recent anthropogenic tain populations, FST estimates between mountain and introductions from similar source populations (Fitz- lowland populations in California exceeded 0.25 in all patrick 1999) or high within-population genetic cases (range = 0.31–0.80), indicating ‘‘very great’’ diversity (e.g., Whitlock and McCauley 1999). Our divergence (Wright 1978). Specifically, the Modern study and Fitzpatrick’s both found substantial genetic Cascades population was highly divergent from all diversity within these recently founded lowland red lowland populations (FST range: 0.56–0.80). Thus, we fox populations, which is consistent with multiple conclude that the modern-day Lassen Peak population introductions from several source populations (Lewis should be managed as the native, state-threatened et al. 1999). Sierra Nevada red fox, in the absence of any evidence Our evidence that the Sacramento Valley population is to the contrary. more similar to the native mountain red fox than to the Also in contrast to the mountain populations, other lowland populations in California contradicts pre- lowland red fox populations in California did not vious ideas about this population. Grinnell et al. (1937: constitute a single interbreeding population. The 385–386) found the presence of red fox in the Sacramento Sacramento Valley population, which is the original Valley ‘‘altogether anomalous’’ considering the boreal lowland population in California (Grinnell et al. habitats favored by the native red fox, leading these au- 1937), was clearly distinct from the other two recently thors to surmise that the population had been ‘‘planted founded lowland populations (Lewis et al. 1999). This there by man’’ by the late 1880s. Their hypothesis was observation supports the previous suggestion (CDFG supported by morphological evidence indicating that the 1999) that the San Francisco Bay Area and Southern Sacramento Valley foxes more closely resembled speci- California populations were not founded by foxes mens from central North America (V. v. regalis)than dispersing from the Sacramento Valley. Three of the specimens from the Sierra Nevada (Roest 1977). How- eight haplotypes in the Bay Area and Southern ever, the fact that the dominant haplotype in the Sacra- California populations (30% and 23% of samples, mento Valley differs by only a single substitution from that respectively) shared the 3 bp signature present in the in the mountain populations, but by up to seven substi- European fox samples but absent from the historic tutions from haplotypes in the other low elevation popu- populations in California’s mountains and Sacra- lations, clearly indicates that the Sacramento Valley foxes mento Valley. The presence of this signature does are more closely related to the native mountain foxes than not necessarily indicate an anthropogenic transloca- to the other lowland populations. Moreover, the occur- tion from Europe to California in the past century, as rence of shared haplotypes between the mountain and none of the complete haplotypes matched any of the Sacramento Valley populations suggests the possibility 17 European haplotypes reported by Frati et al. that the observed differences in haplotype frequencies (1998). It is possible that these haplotypes evolved in could have arisen solely via drift over the past century.

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Threats of hybridization vational stratification of the two species in regions of Alberta (Dekker 1989) and Maine (Fuller and Harri- It has been hypothesized that mountain red fox are son 2006) and possibly the Lassen Peak region of specialists that became restricted to high-elevation California (Perrine 2005). However, coyotes and red habitats following the retreat of the glaciers at the end of foxes co-occur at lower elevations in California (e.g., the Pleistocene, and that lowland red fox are dietary and Ralls and White 1995), and there is no direct evidence habitat generalists that could potentially disperse into that the presence of coyotes is the primary factor these mountainous areas and threaten the persistence of separating lowland from mountain red fox populations. mountain red fox via hybridization and resource com- The available genetic evidence suggests that the barrier petition (e.g., Aubry 1983; Lewis et al. 1995; Kamler and between mountain and lowland red fox populations has Ballard 2002). Of all the lowland populations repre- existed for more than a century, although the mecha- senting such a threat to the Lassen Peak population, the nisms that created and maintain it are unclear. Sacramento Valley region is the closest geographically, Our results underscore the need for several further and therefore the most likely source of lowland immi- investigations. Analyses employing nuclear markers are gration. However, the pairwise FST value between the needed to quantify the extent of male-biased gene flow Lassen region and the modern Sacramento Valley between the lowlands and the mountains, and to detect population was the largest of any in this study, indicating additional private alleles within these populations. low or nonexistent gene flow. It is unclear what has Incorporating historic and modern specimens from the prevented substantial immigration from the valley to Cascade Range and Rocky Mountains could resolve the mountains, or vice versa, over the past century. Red longstanding questions about the shared origin of these foxes are mobile and highly adaptable generalists, and three mountain subspecies (Aubry 1983) and indicate few barriers to dispersal have been identified other than whether there is genetic support for the proposal that major rivers and water courses (Storm et al. 1976; Voigt they be relegated to a single subspecies (Roest 1979). 1987). Yet, similar to California, an unidentified barrier Likewise, the acquisition of additional lowland speci- exists between mid-elevation and high-elevation red fox mens from throughout California, available due to populations in Yellowstone National Park (Swanson control operations to protect native species, could more et al. 2005). In the Cascades of Washington, a dense belt fully elucidate current patterns of gene flow and genetic of conifer forest separates the lowland red fox from the structure among these populations. Determining the native mountain red fox, but the forest itself is probably origins of the lowland populations, especially the Sac- not the barrier to gene flow as this habitat could easily be ramento Valley population, would require incorporat- crossed by red foxes (Aubry 1984). ing samples from a broader geographic area to include The barrier restricting mountain red foxes to high multiple potential source populations and utilizing elevation habitats may also prevent lowland red foxes higher-resolution markers such as the mitochondrial from dispersing into or establishing there. For exam- control region. ple, dispersing red foxes may select habitats that are similar to their natal habitats, as has been hypothesized Management implications to account for habitat-specific population structure in coyotes (Canis latrans; Sacks et al. 2004, 2005) and gray Several western states, including California, Oregon, wolves (Canis lupus; Carmichael et al. 2001; Geffen Washington, Utah, and Idaho, likely host both native et al. 2004). Habitat-specific behavioral differences, mountain and exotic lowland red fox populations (Au- including differences in social structure, dispersal rates bry 1984; Kamler and Ballard 2002). However, only and dispersal distances, likely contribute to the signif- California has separate management strategies for icant genetic structure observed among urban red fox mountain and lowland populations, under the assump- populations and between the urban and adjacent rural tion that one is native and the other exotic (Kamler and populations (Robinson and Marks 2001; Simonsen Ballard 2002). Although the elevation boundary of et al. 2003; Wandeler et al. 2003a). Alternatively, the 1066 m (3500 ft) used to delimit these populations in barrier could be extrinsic, such as the presence of California is arbitrary and the true boundary likely coyotes or other dominant competitors between the varies with latitude, our findings indicate that this mountain and lowland red fox populations. Coyotes operational criterion successfully separates high-eleva- can be an important source of mortality for smaller tion from lowland populations. Unfortunately, no canids (Sargeant and Allen 1989; Ralls and White 1995; specimens from the Sierra Nevada have been collected Palomares and Caro 1999; Farias et al. 2005). Spatial since 1941. In fact, it is unclear whether any red foxes avoidance of coyotes by red foxes has led to the ele- currently inhabit the Sierra Nevada. Recent surveys

123 Conserv Genet using baited camera traps and track plates failed to de- threat of future immigration from lowland red fox tect red fox anywhere in the Sierra Nevada (Zielinski populations, either from the west (Lewis et al. 1995)or et al. 2005), including historic population centers such as from the east (Kamler and Ballard 2002), should not be Sequoia and Kings Canyon National Parks (Green discounted. The distribution and range expansion of 2006). Additional targeted surveys are necessary in these exotic populations should be carefully moni- these areas, preferably involving the collection of ge- tored, not just for the beneﬁt of the native red fox but netic samples via hair snares and the collection of feces. also for numerous other native species that may be Collaborative efforts with state and federal agencies negatively impacted (Lewis et al. 1999). should be established so that any available specimens (e.g., road-kills) within the historic range of the Sierra Acknowledgements The following collection managers made Nevada red fox are collected for analysis. specimens available for this study: J. Bogiatto (California State University, Chico), P. Collins (Santa Barbara Natural History The distribution of mountain red fox appears to be Museum), C. Conroy and E. Lacey (Museum of Vertebrate Zool- extremely limited in California and their population ogy), J. Dines and I. Horovitz (Natural History Museum of Los density and distribution appear to have declined con- Angeles County), and T. Schweitzer (Fort Roosevelt Vertebrate siderably in recent decades. We found no evidence that Collection). Special thanks to M. Schwartz and two anonymous reviewers whose comments greatly improved the manuscript. this decline is due to competition with or displacement K. Aubry, S. Beissinger, A. Bidlack, C. Epps, B. Lidicker, P. Pals- by the exotic red fox; however, without evidence from bøll, J. Patton, M. Statham and S. Wisely provided additional nuclear markers, genetic introgression cannot be ruled manuscript comments and suggestions. This research was supported out. Even if hybridization has not occurred to date, the in part by the USDI National Park Service’s Biological Resource Management Division and Lassen Volcanic National Park. Appendix

Table 4 Red fox specimens obtained for this study (n = 85) Population California county Sample ID Year Collection Institution PCR elevation (m)

Historic CS Lassen MVZ-34984 1925 1850 MVZ OK Lassen MVZ-35280 1925 1850 MVZ OK Siskiyou MVZ-3296 1904 2135 MVZ Fail Siskiyou MVZ-68857 1934 2050 MVZ OK Siskiyou MVZ-68858 1934 2050 MVZ OK Historic SN Mariposa MVZ-23696 1916 1350 MVZ OK Mono MVZ-32800 1921 2950 MVZ OK Mono MVZ-32809 1922 3000 MVZ OK Mono MVZ-33381 1922 3050 MVZ OK Mono MVZ-33382 1922 3050 MVZ OK Mono MVZ-33472 1923 3000 MVZ OK Mono MVZ-33473 1923 3000 MVZ OK Mono MVZ-33474 1923 3050 MVZ OK Mono MVZ-33586 1923 3100 MVZ OK Mono MVZ-33587 1923 3100 MVZ OK Mono MVZ-41004 1928 2950 MVZ OK Mono MVZ-41468 1928 2950 MVZ OK Mono MVZ-44097 1929 2950 MVZ OK Mono MVZ-46663 1929 2950 MVZ OK Nevada MVZ-95401 1941 1850 MVZ OK Tulare MVZ-16251 1911 3000 MVZ OK Tulare MVZ-16252 1911 3000 MVZ OK Tulare MVZ-16374 1911 2450 MVZ OK Historic SV Colusa MVZ-33550 1923 <50 MVZ OK Colusa MVZ-36492 1926 <50 MVZ OK Colusa MVZ-36493 1926 <50 MVZ Fail** Colusa MVZ-36494 1926 <50 MVZ OK Colusa MVZ-36495 1926 <50 MVZ OK** Colusa MVZ-36496 1926 <50 MVZ OK** Colusa MVZ-36497 1926 <50 MVZ OK Colusa MVZ-46865 1926 <50 MVZ Fail** Colusa MVZ-70285 1935 <50 MVZ OK Glenn MVZ-44095 1929 <50 MVZ OK Tehama MVZ-115439 1948 50 MVZ OK

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Table 4 continued Population California county Sample ID Year Collection Institution PCR elevation (m)

Modern BA Alameda LF-42 * 1995 350 MVZ OK Alameda REJ-1535 1996 50 MVZ OK Alameda REJ-1537 1996 50 MVZ OK Alameda REJ-1540 1996 50 MVZ OK Marin MVZ-175993 1982 <50 MVZ OK San Joaquin REJ-1624 1997 <50 MVZ OK San Mateo REJ-1555 1996 100 MVZ OK San Mateo REJ-1573 1996 100 MVZ OK San Mateo REJ-1575 1996 100 MVZ OK San Mateo REJ-1588 1997 50 MVZ OK Modern CS Tehama F01 1998 1900 J. Perrine OK Tehama F02 2000 1700 J. Perrine OK Shasta F03 2000 2550 J. Perrine OK Tehama F05 2000 1750 J. Perrine OK Tehama M01 1998 1750 J. Perrine OK Shasta Scat 8 1999 2600 J. Perrine Fail Tehama Scat 12 1999 2100 J. Perrine OK Shasta Scat 16 1999 2550 J. Perrine OK Tehama Scat 17 1999 2050 J. Perrine OK Shasta Scat 21 1998 2650 J. Perrine OK Tehama Scat 40 1998 2100 J. Perrine Fail Shasta Scat 330 2001 2050 J. Perrine Fail Shasta Scat 333 2001 2050 J. Perrine Fail Shasta Scat 404 2001 2000 J. Perrine Fail Modern SC Fesno FRC-027 1997 <200 FRVC OK Kern FRC-061 1999 <200 FRVC OK Kern R003 * 2003 50–150 MVZ OK Kings FRC-087 2000 <200 FRVC OK Los Angeles LA-85700 1988 250–400 LA NHM Fail Los Angeles LA-87636 1990 250–400 LA NHM OK Los Angeles LA-5 * 2002 <150 LA NHM OK Los Angeles LA-87624 1989 <150 LA NHM OK Santa Barbara SB-1 * 2002 <150 SB MNH OK Santa Barbara SB-2 * 1993 <150 SB MNH OK Santa Barbara SB-3 * 1990 <150 SB MNH OK Santa Barbara SB-4 * 1994 <150 SB MNH OK Santa Barbara SB-5 * 1995 <150 SB MNH OK Santa Barbara SB-6 * 1996 <150 SB MNH OK Modern SV Butte CSU-2530 1966 <100 CSUC OK Butte CSU-3943 1970 <150 CSUC OK Butte CSU-1 * 1995 <100 CSUC Fail Butte X-2 2001 <50 R. Alessio OK Colusa CSU-2588 1968 <50 CSUC OK Colusa X-1 2000 <50 J. Perrine OK Glenn CSU-2589 1969 <100 CSUC OK Glenn CSU-5128 1973 <100 CSUC OK Glenn PW-1 2004 100 P. Weliver OK Napa CSU-2591 1968 <350 CSUC OK Tehama CSU-3504 1970 <100 CSUC OK Tehama H-1 * 1986 <350 HSU OK Yolo W-1 2002 50 J. Perrine OK Collection elevations rounded to the nearest 50 m. Institution abbreviations are as follows: CSUC = California State University, Chico; FRVC = Fort Roosevelt Vertebrate Collection, Hanford; HSU = Humboldt State University, Arcata; LA NMH = Los Angeles County Natural History Museum; MVZ = Museum of Vertebrate Zoology, UC Berkeley; SB NHM = Santa Barbara Natural History Museum. Other names indicate specimens not from official collections, such as field study animals or roadkills. PCR indicates whether a specimen yielded a usable sequence (‘‘OK’’; n = 75) or not (‘‘fail’’; n = 10; also includes scats that could not be reliably differentiated from other individuals via microsatellites) * specimen not yet assigned an official number by the institution ** specimen excluded from analysis because the animal was a known littermate of another specimen yielding a usable sequence

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